Project Summary Horizontal gene transfer (HGT) in the human microbiome has considerable impact on human health through its role in disseminating virulence factors and antibiotic resistance. Due to the complexity of studying HGT within microbiomes, however, it has received limited experimental attention. This proposal characterizes dynamics of HGT in the gut microbiome through the development and application of new experimental tools. We will begin by optimizing a technique developed in our preliminary work called ET-Seq, which serves as a sequence-based tool to measure permissiveness of microbial community members to HGT agents of interest. The approach will be applied to test the ability of human microbiota members to take up DNA from various forms of HGT. Agents for conjugation, transduction, and transformation will be assayed in parallel by ET-Seq for their efficiency against the microbial constituents of a realistic in vitro model of the human gut microbiome. Using comparative genomics, the community landscape of permissiveness and restrictiveness to the different means of HGT will be linked to the genes responsible. We will also supplement ET-Seq's static snapshot of the state of HGT by creating molecular recordings of HGT. To accomplish this, we will add CRISPR-Cas based molecular recorders to HGT agents so that they record their trajectory through the gut microbiota by picking up host DNA as they travel. These routes of HGT will later be recovered by sequencing of the HGT agent's recording cassettes. Together these approaches will provide a dynamic and comprehensive view of HGT in the human gut microbiome. This work has potential for far reaching human health impacts. HGT is extremely prevalent in the gut and serves to spread drug resistance and virulence genes. Understanding how HGT occurs should provide data necessary to mitigate this subset of undesirable events. Furthermore, this work will provide a foundation for targeted editing within the gut microbiome. It will elucidate the effectiveness of various gene transfer agents on gut microbiota members and provide the tools necessary to track genetic changes implemented in this, or any other community. The computational and molecular ecology skillsets of Jill Banfield's lab, the molecular biology and CRISPR-Cas expertise of Jennifer Doudna's lab, and the gut microbiome models of Melanie Ott's lab will provide broad resources for the success of this project as well as extensive training potential.